Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention relates to the field of monoclonal antibody production.
• mAbs Monoclonal antibodies
• MAbs are an important class of biopharmaceuticals. They represent one of the best selling classes of biologies, with combined US sales reaching about $16.9 billion in 2009 (Aggarwal, 2010).
• MAbs are commonly used as diagnostic agents and as drugs, especially for treatment of various types of cancers and chronic inflammatory conditions.
• mAbs offer patients many new treatment options that are more effective, safer and more convenient than other traditional treatments (Jain & Kumar, 2008).
• DLL4 human delta-like antigen 4
• Several human monoclonal antibodies specific for DLL4 are described in U.S. patent application no. 2010/01963850.
• MAbs for therapeutic applications can be expressed using Chinese Hamster Ovary ("CHO") cells. Genes encoding such mAbs can be cloned and transfected into a CHO cell line which permits production of sufficient quantities of the mAb for clinical and commercial use. CHO cell clones transfected with the genes encoding the mAbs, upon expression and protein affinity ⁇ e.g., protein A) purification, may yield unacceptably high levels of antibody aggregate.
• CHO Chinese Hamster Ovary
• MAb aggregation is a major concern in therapeutic protein production.
• WHO World Health Organization
• WHO World Health Organization
• aggregates reduce product purity and quality. They may cause an immunogenic response in patients (Barnard et al., 2010).
• aggregates may mechanically block capillaries causing reduced microcirculation in postischemic patients (Shellekens, 2005; Rosenberg, 2006). Accordingly, if aggregates cannot be reduced to an acceptable level below the WHO limits, an antibody with therapeutic potential may be dropped from development.
• Aggregates may form at any step in the manufacturing process, including during culture of mammalian, e.g., CHO, cells. If aggregation for a mAb could be reduced at the cell culture stage, this would be highly beneficial as it would significantly reduce the downstream manufacturing burden and result in substantial overall process yield improvement.
• the fermentation process can yield a protein A-purified product from the culture having an aggregation level less than 5%.
• an embodiment encompassed by the invention provides a method of producing an anti-DLL4 monoclonal antibody.
• Mammalian cells that express the monoclonal antibody are cultured at a temperature of about 36.5 °C, a pH of about 6.85, and a starting osmolality of about 320mOsm/kg H 2 0.
• mammalian cells that express the monoclonal antibody are cultured at a temperature of about 37°C, a pH of about 7.0, and a starting osmolality of about 320mOsm/kg H 2 0.
• the expressed antibody is recovered from the culture supernatant.
• Another embodiment encompassed by the invention is a method of reducing aggregates of an anti-DLL4 monoclonal antibody.
• a CHO cell that secretes the anti-DLL4 monoclonal antibody is cultured under conditions of temperature, pH, and osmolality that produce less aggregate than culture of the same mAb-producing CHO cell under conditions comprising a temperature of 36.5°C, a pH of 6.8, and a starting osmolality of 320 mOsm/kg H 2 O in a bioreactor.
• the anti-DLL4 monoclonal antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1 , a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.
• the method can comprise a multi-part feed.
• the multi-part feed can comprise a two-part feed.
• the method can comprise a single feed.
• Glucose can be added to the culture to control the glucose level. Where glucose is added in the method, the addition of glucose is not considered a feed.
• FIG 1 presents an HPLC profile showing antibody monomer and aggregate peaks.
• FIG 2 presents aggregate levels compared between twelve 1 L bioreactors with different cell culture conditions.
• FIG 3A- 3F present a Design of Experiments (DoE) prediction profiler showing the relationship between temperature, osmolality, and pH as cell culture parameters in a linear model, and their impacts on titer and aggregate levels (post Protein A purification).
• DoE Design of Experiments
• FIG 4 presents the cell growth pattern observed in previous (red/bottom line) and new optimised (green/top line) fermentation process.
• FIG 5 presents a Design of Experiments Contour Profiler presenting the operating window for the cell culture process to achieve the aggregation level below 2% and titre above 6 g/L without reaching peak viable cell number higher than 24 x 10 6 viable cells/mL.
• FIG 6 presents contour plots showing the relationship between temperature, osmolality, and pH as cell culture parameters in a quadratic model, and their impacts on titer and aggregate levels (post Protein A purification). Contour plots show the titre (top plots) and aggregate (bottom plots) range predictions as a function of pH (X axis), Temperature (Y axis) and osmolality (indicated above the plots).
• the disclosure provides a method of reducing aggregates of an anti-DLL4 monoclonal antibody (mAb) recovered from cell culture or from affinity-purified cell culture supernatant.
• the aggregates may also be reduced by culturing a first mammalian cell line that secretes the mAb under conditions of temperature, pH, and osmolality that produce less aggregate than culture of the same mammalian cell line that secretes the mAb under conditions including a temperature of 36.5°C, a pH of 6.8, and starting osmolality of 320 mOsm/kg H 2 O in a bioreactor using a single feed.
• a second mammalian cell line that produces lower levels of mAb aggregate than the first mammalian cell line may be selected and substituted for the first mammalian cell line.
• the pH, temperature, and starting osmolality culture conditions that reduce the aggregates of the anti-DLL4 mAb may be: pH 7.0, temperature 34 °C, and starting osmolality 400 mOsm/kg H 2 O; or pH 6.85, temperature 35.5°C, and starting osmolality 360 mOsm/kg H 2 O; or pH 6.7, temperature 37°C, and starting osmolality 400 mOsm/kg H 2 0; or pH 6.7, temperature 34 °C, and starting osmolality 320 mOsm/kg H 2 0; or pH 7.0, temperature 37°C, and starting osmolality 320 mOsm/kg H 2 0; or pH 7.0, temperature 37°C, and starting osmolality 400 mOsm/kg H 2 0; or pH 6.85, temperature 35.5°C, and starting osmolality 360 mOsm/kg H 2 0; or pH 7.0,
• the pH, temperature, and starting osmolality culture conditions that reduce the aggregates of the anti-DLL4 mAb to 5% or less may be a pH of between about 6.80 and about 7.00, osmolality of between about 320 and about 400 mOsm/kg H 2 0, and a temperature between about 34.5 °C and about 36.5°C.
• the anti-DLL4 mAb may be recovered from the cell culture following clarification and/or may be affinity purified utilizing, e.g., protein A affinity chromatography.
• the culture temperature may be 37°C, or may be about 36.5°C. In one embodiment, the culture temperature is about 37.0 °C. In another embodiment, the culture temperature is held at 37.0 °C within the margin of error of a bioreactor culture system. In yet another embodiment, the culture temperature is held at 37.0 °C within the margin of error of a DASGIP 1 L fed-batch bioreactor. In still other embodiments, the culture temperature may be 37.0°C ⁇ 0.1 . In one embodiment, the culture temperature is 37°C.
• the culture temperature may be 36.5°C, or may be about 36.5°C. In one embodiment, the culture temperature is about 36.5°C. In another embodiment, the culture temperature is held at 36.5°C within the margin of error of a bioreactor culture system. In yet another embodiment, the culture temperature is held at 36.5°C within the margin of error of a DASGIP 1 L fed-batch bioreactor. In still other embodiments, the culture temperature may be 36.5°C ⁇ 0.1 . In one embodiment, the culture temperature is 36.5°C.
• the culture pH is about 7.0. In one embodiment, the culture pH is held at 7.0 within the margin of error of a bioreactor culture system. In another embodiment, the culture pH is held at 7.0 ⁇ 0.1 in a DASGIP 1 L fed-batch bioreactor. In one embodiment, the culture pH is 7.0. In some embodiments of the various aspects disclosed herein, the culture pH is about 6.85. In one embodiment, the culture pH is held at 6.85 within the margin of error of a bioreactor culture system. In another embodiment, the culture pH is held at 6.85 ⁇ 0.1 in a DASGIP 1 L fed-batch bioreactor. In one embodiment, the culture pH is 6.85. The pH may be adjusted during the culture process, for example, by adding alkali solution, sodium bicarbonate or C0 2 gas.
• the osmolality of the culture medium at the start of culture is about 320 mOsm/kg H 2 0.
• the osmolality of the culture medium at the start of culture is 320 mOsm/kg H 2 0 ⁇ 1 .0.
• the osmolality of the culture medium at the start of culture is 320 mOsm/kg H 2 0.
• the culture medium is an animal protein-free medium. Osmolality of the medium can be adjusted, for example, by adding a salt such as NaCI.
• the culture medium may be any well known in the art or may be a media custom made by the user.
• the culture process may include a 2-part feed.
• the feed is present as a two- part (i.e., stored in 2 separate containers) concentrate and each part of the concentrate is added individually to the culture.
• feeds may contain media or grouped media components such as amino acids, vitamins, iron, lipids, and trace elements. Feeds may be delivered to cells in one- part, or two-parts, or three-parts, or more parts.
• Commercially available feeds include IS CHO FEED CDTM (Irvine Scientific) and CHO CD EfficientFeedTM (Invitrogen).
• an animal protein-free medium with starting osmolality of 320 mOsm/kg H 2 O is used to culture mAb-secreting cells in a bioreactor where the temperature is set to 37.0 °C and the pH is held at 7.0 ⁇ 0.1 during the culture, the culture process includes a 2-part feed.
• an animal protein-free medium with starting osmolality of 320 mOsm/kg H 2 O is used to culture mAb-secreting cells in a bioreactor where the temperature is set to 36.50 °C and the pH is held at 6.85 ⁇ 0.1 during the culture, the culture process includes a 2-part feed.
• the method reduces the percentage of aggregates of the anti-DLL4 mAb in the supernatant of cultured cells expressing the anti-DLL4 mAb and the percentage anti-DLL4 mAb aggregate reduction may be measured in a sample of the cell culture supernatant, or in in a sample of the cell culture supernatant following clarification, or in a sample of the cell culture supernatant following clarification and affinity purification.
• the reduction of the percentage of aggregates of the anti-DLL4 antibody may be a reduction of aggregates recovered in supernatant from the cells following clarification and the percentage aggregates may be determined from a sample of the clarified cell culture supernatant or may be determined from a sample of the clarified cell culture supernatant following affinity purification.
• the reduction of aggregates of the anti-DLL4 antibody may be a reduction of aggregates recovered from affinity-purified cell culture supernatant of the cells expressing the DLL4 mAb.
• the percentage aggregates may be determined from a sample of the affinity-purified cell culture supernatant.
• the supernatant of the cells expressing the mAb or clarified supernatant of the cells expressing the mAb may be affinity purified.
• affinity purification methods include, without limitation, purification by Protein A, Protein G, Protein A/G, or Protein L affinity chromatography.
• affinity chromatography methods include those which employ immobilized antigen, i.e., DLL4, to which the mAb specifically binds.
• Another aspect of the disclosure provides a method of reducing aggregate content in a protein A-purified mAb product to less than about 5%.
• the method comprises steps of: culturing a mammalian cell line that expresses an anti-DLL4 mAb in a culture medium having a starting osmolality of about 320 mOsm/kg H 2 0, at a temperature of about 37 °C, and at a pH of about 7.0.
• Another aspect of the disclosure provides a method of reducing aggregate content in a protein A-purified mAb product to less than about 5%.
• the method comprises steps of: culturing a mammalian cell line that expresses an anti-DLL4 mAb in a culture medium having a starting osmolality of about 320 mOsm/kg H 2 0, at a temperature of about 36.5 °C, and at a pH of about 6.85.
• the mammalian cell line expresses an anti-DLL4 antibody comprising a heavy chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a light chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
• the culturing process comprises utilizing a two part feed to feed the cells during the culturing to thereby reduce aggregate formation in the culture supernatant.
• the antibody expressed by the mammalian cell line is then recovered from the culture supernatant.
• the antibody may be purified using an affinity chromatography step, e.g., protein A.
• the aggregate content is measured by HPLC-SEC. In one embodiment, the aggregate content is less than about 5%, about 4%, about 3%, or about 2%. In one embodiment, the aggregate content is about 1 .2%, about 1 .3%, about 1 .4%, about 1 .5%, about 1.6%, about 1 .7%, about 1 .8%, or about 1 .9%.
• the disclosure also provides a method of producing an anti-DLL4 monoclonal antibody comprising steps of: culturing a Chinese Hamster Ovary (CHO) cell that expresses an antibody heavy chain variable domain as set forth in SEQ ID NO: 7 and a light chain variable domain as set forth in SEQ ID NO: 8 in a culture medium with a starting osmolality of about 320 mOsm/kg H 2 0, at a temperature of about 37 °C, and a pH of about 7; using a two part feed to feed the cells during the culturing process; and recovering the expressed anti-DLL4 antibody from the culture supernatant.
• CHO Chinese Hamster Ovary
• the disclosure also provides a method of producing an anti-DLL4 monoclonal antibody comprising steps of: culturing a Chinese Hamster Ovary (CHO) cell that expresses an antibody heavy chain variable domain as set forth in SEQ ID NO: 7 and a light chain variable domain as set forth in SEQ ID NO: 8 in a culture medium with a starting osmolality of about 320 mOsm/kg H 2 0, at a temperature of about 36.5 °C, and a pH of about 6.85; using a two part feed to feed the cells during the culturing process; and recovering the expressed anti-DLL4 antibody from the culture supernatant.
• CHO Chinese Hamster Ovary
• the expressed antibody may be recovered from the culture supernatant by protein A chromatography.
• the disclosure also provides for a method of producing an anti-DLL4 monoclonal antibody, comprising:
• the anti-DLL4 antibody comprises a heavy chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a light chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and wherein the culture medium has a starting osmolality of about 320 mOsm/kg H 2 0;
• the disclosure also provides for a method of producing an anti-DLL4 monoclonal antibody, comprising:
• the anti-DLL4 antibody comprises a heavy chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a light chain variable domain comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and wherein the culture medium has a starting osmolality of about 320 mOsm/kg H 2 O;
• the disclosure also provides a method of producing an anti-DLL4 monoclonal antibody comprising: culturing a Chinese Hamster Ovary (CHO) cell that expresses the antibody heavy and light chains in a culture medium with a starting osmolality of about 320 mOsm/kg H 2 0, at a temperature of about 37°C and a pH of about 7.0, wherein the CHO cell is fed during the culturing process using a 2 part feed; and recovering the antibody from the culture supernatant.
• CHO Chinese Hamster Ovary
• the disclosure also provides a method of producing an anti-DLL4 monoclonal antibody comprising: culturing a Chinese Hamster Ovary (CHO) cell that expresses the antibody heavy and light chains in a culture medium with a starting osmolality of about 320 mOsm/kg H 2 O, at a temperature of about 36.5°C and a pH of about 6.85, wherein the CHO cell is fed during the culturing process using a 2 part feed; and recovering the antibody from the culture supernatant.
• CHO Chinese Hamster Ovary
• the recovery step may comprise purifying the mAb on protein A.
• the protein A-purified mAb may have an aggregate content of less than about 5%, about 4%, about 3%, or about 2%.
• the protein A-purified mAb has an aggregate content of about 1 .2%, about 1 .3%, about 1 .4%, about 1 .5%, about 1 .6%, about 1 .7%, about 1.8%, or about 1 .9%.
• the mammalian cell line that expresses an antibody may be chosen from Chinese hamster ovary (CHO) cells, NS0 cells, or PER.C6 (ECACC no. 96022940) cells.
• the mammalian cell line is CHO.
• the CHO cell line may be CHOK1 SV cells (Lonza).
• the cell line that secretes a mAb may be transfected with an appropriate gene or genes that express the mAb.
• percentages of aggregate or a percentage of monomer in a mAb product may be determined utilizing techniques such as field-flow fractionation, analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, or other methods known in the art. For example, percentage may be determined employing HPLC-SEC analysis of protein A purified mAb samples to quantitate amounts of monomer and aggregates in the total mAb amount.
• the disclosure also provides for an anti-DLL4 mAb produced according to any of the described culture methods.
• the disclosure further provides for a composition comprising the DLL4 mAb produced according to of the methods.
• the anti-DLL4 mAb product or composition may comprise less than about 1 .4% aggregate following protein A purification. The percentage of aggregate may be determined by HPLC-SEC.
• composition comprising, consisting essentially of, or consisting of an anti-DLL4 mAb produced by a method as described herein and a pharmaceutically acceptable carrier.
• the anti-DLL4 mAb may be a mAb that is a human lgG1 mAb that binds DLL4.
• the mAb may be an anti-DLL4 antibody that comprises a variable heavy chain amino acid sequence comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO:1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a variable light chain amino acid sequence comprising CDR1 , CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
• the mAb may be an anti-DLL4 antibody that comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO: 7.
• the mAb may be an anti-DLL4 antibody that comprises a light chain polypeptide comprising the sequence of SEQ ID NO:8.
• the mAb may be an anti-DLL4 antibody that comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO:7 and a light chain polypeptide comprising the sequence of SEQ ID NO:8.
• the anti-DLL4 mAb may be a fully human monoclonal antibody.
• the mAb is an anti-DLL4 antibody that comprises a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three of the CDRs of the antibody encoded by the polynucleotide in the plasmid designated Mab21 H3VH, which was deposited at the American Type Culture Collection (ATCC) under number PTA-9501 on Sep. 17, 2008.
• ATCC American Type Culture Collection
• the mAb is an anti-DLL4 antibody that comprises a variable light chain amino acid sequence comprising at least one, at least two, or at least three of the CDRs of the antibody encoded by the polynucleotide in the plasmid designated Mab21 H3VLOP, which was deposited at the ATCC under number PTA-9500 on Sep. 17, 2008.
• the mAb is an anti-DLL4 antibody that comprises a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three of the CDRs of the antibody encoded by the polynucleotide in the plasmid designated Mab21 H3VH, which was deposited at the ATCC under number PTA-9501 on Sep. 17, 2008; and comprises a variable light chain amino acid sequence comprising at least one, at least two, or at least three of the CDRs of the antibody encoded by the polynucleotide in the plasmid designated Mab21 H3VLOP, which was deposited at the ATCC under number PTA-9500 on Sep. 17, 2008.
• the mAb may be an anti-DLL4 antibody that comprises all three heavy chain CDR amino acid sequences encoded by the polynucleotide in the plasmid designated Mab21 H3VH, which was deposited at the ATCC under number PTA-9501 on Sep. 17, 2008, and all three light chain CDR amino acid sequences encoded by the polynucleotide in the plasmid designated Mab21 VLOP, which as deposited at the ATCC under number PTA-9500 on Sep. 17, 2008.
• the mAb may be an anti-DLL4 antibody that comprises the heavy chain amino acid sequence encoded by the polynucleotide in the plasmid designated Mab21 H3VH, which was deposited at the ATCC under number PTA-9501 on Sep. 17, 2008, and the light chain amino acid sequence encoded by the polynucleotide in the plasmid designated Mab21 VLOP, which as deposited at the ATCC under number PTA- 9500 on Sep. 17, 2008.
• the various methods of reducing aggregates of an anti-DLL4 monoclonal antibody (mAb) recovered from cell culture or from affinity-purified cell culture supernatant may further result in increased mAb titer.
• the methods may increase mAb titre by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, or by at least 200%.
• the mAb titer may be at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, at least at least 5 g/L, at least 5.5 g/L, at least 6 g/L, at least 6.25 g/L, at least 6.5 g/L, at least 6.75 g/L, at least 7 g/L, at least 7.25 g/L, at least 7.5 g/L, at least 7.75 g/L, at least 8 g/L or at least 8.5 g/L.
• Standard techniques are typically used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001 )), which is incorporated herein by reference.
• antibody refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen chain.
• Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
• Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
• Light chains are classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain constant region.
• the variable domain of a kappa light chain may also be denoted herein as VK.
• the term "variable region" may also be used to describe the variable domain of a heavy chain or light chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
• the variable regions of each light/heavy chain pair form an antibody binding site.
• Such antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs,
• Antibodies include immunoglobulin molecules, i.e., molecules that contain an antigen-binding site.
• Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
• a "human antibody” is an antibody derived from a human or an antibody obtained from a transgenic organism that has been engineered to produce human antibodies in response to antigenic challenge.
• a human antibody may also include an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.
• a fully human antibody can be constructed by genetic or chromosomal transfection methods, phage display technology ⁇ e.g., US Patent No. 5,969,108), or in vitro activated B cells ⁇ e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).
• An antibody may be from any species.
• mAb refers to a monoclonal antibody.
• a monoclonal antibody is an antibody derived from a single cellular source, such as a hybridoma, a transformed cell, or a cell made to express the genes encoding an antibody by transfection or other technique.
• Suitable mammalian cell lines include Chinese Hamster Ovary ("CHO"), NS0, or PER.C6 (ECACC no. 96022940) cell lines. Any of these mammalian cell lines can be generally transfected with one or more recombinant vectors that encode the heavy and light chains, or fragments thereof, of a mAb of interest. The transfected cells secrete a mAb comprising the encoded heavy and light chains into the cell culture medium (supernatant).
• Examples of mAbs suitable for use in the methods and compositions of the disclosure are the human anti-DLL4 antibodies described in US2010/0196385 or WO 2010/032060, each of which are incorporated by reference.
• an anti-DLL4 mAb suitable for use in the methods and compositions provided herein comprises a heavy chain variable domain comprising CDR1 : NYGIT (SEQ ID NO:1 ); CDR2: WISAYNGNTNYAQKLQD (SEQ ID NO:2); and CDR3: DRVPRIPVTTEAFDI (SEQ ID NO: 3), and a light chain variable domain comprising CDR1 : SGSSSNIGSYFVY (SEQ ID NO:4); CDR2: RNNQRPS (SEQ ID NO;5); and CDR3: AAWDDSLSGHWV (SEQ ID NO: 6).
• an anti-DLL4 mAb suitable for use in the methods and compositions provided herein comprises a heavy chain variable domain comprising CDR1 : NYGIT (SEQ ID NO:1 ); CDR2: WISAYNGNTNYAQKLQD (SEQ ID NO:2); and CDR3: DRVPRIPVTTEAFDI (SEQ ID NO: 3
• DLL4 refers to the molecule that is DLL4 protein, also known as Delta-like protein 4 precursor, Drosophila Delta homolog 4, hdelta2, MGC126344, or UNQ1895/PR04341 .
• binding fragment(s) includes single-chain Fvs (scFv), single- chain antibodies, single domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab') 2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-stabilised variable region (dsFv), dimeric variable region (Diabody), anti-idiotypic (anti-Id) antibodies, intrabodies, linear antibodies, single- chain antibody molecules and multispecific antibodies formed from antibody fragments and epitope-binding fragments of any of the above.
• Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
• Antibodies as described herein, can be prepared in a mixture with a pharmaceutically acceptable carrier.
• Embodiments of the invention include sterile pharmaceutical formulations of antibodies.
• Sterile formulations can be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution of the antibody.
• Antibodies ordinarily will be stored in lyophilized form or in solution.
• Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
• a "therapeutically effective" amount is an amount that provides some improvement or benefit to the subject.
• a “therapeutically effective” amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom. Further, those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
• clone 31 -121 produced particularly high levels of aggregate. Like the other clones, it comprises a heavy chain variable domain as set forth in SEQ ID NO: 7 and a light chain variable domain as set forth in SEQ ID NO: 8. It has an isometric point of 9.0 and as a monomer is 148 kDa.
• Cell culture maintenance Clone 31 -121 cells were maintained in 250 ml_ shake flasks containing 50 ml_ of animal protein free medium (M18). Cells were seeded at a cell seeding of 3 x 10 5 viable cells/mL in in-house animal protein-free medium (M18) supplemented with glutamine synthetase inhibitor L-methionine sulfoximine (MSX). Cell cultures were maintained under continuous shaking at 140 rpm in an atmosphere of 5% of C02/95% air at 36.5 °C and passaged every three days.
• M18 animal protein free medium
• MSX glutamine synthetase inhibitor L-methionine sulfoximine
• Cell culture processes in 1 L Bioreactors Cell culture processes were carried out in two blocks of six 1 L fed-batch bioreactors (DASGIP AG, Julich, Germany). The cells were seeded at a cell density between 8 and 10 x 10 5 viable cells/mL in M18 medium without any supplements. Cell cultures were maintained under continuous stirring of 150 rpm and then 175 rpm from day 7. Dissolved oxygen, pH and temperature were measured online using appropriate probes. This information was used to activate oxygen sparging to maintain dissolved oxygen, activate C0 2 sparging or pumping of alkali to maintain pH (with a deadband of 0.1 pH unit) and activate a heating blanket to maintain temperature.
• DASGIP AG 1 L fed-batch bioreactors
• the protein A purification was performed using MabSelect SuRe (GE Healthcare, Uppsala, Sweden), packed into a Vantage-L 1 1 column (Millipore, MA, USA).
• the MabSelect SuRe column was equilibrated with phosphate buffered saline and was then loaded with clarified cell culture supernatant, to a capacity of 30 g mAb per liter of resin.
• the column was then subjected to a requilibration and two wash steps before being eluted at low pH. All Protein A purification runs were performed using an AKTA van controlled using Unicorn 6 software (both from GE Healthcare, Uppsala, Sweden).
• SEC Size Exclusion Chromatography
• Elution was performed using 0.1 M sodium phosphate and 0.1 M sodium sulphate buffer at pH 6.8 at flow rate of 1.0 mL/min for chromatographic separation on a HPLC system. Prior to use, the column was calibrated using BioRad gel filtration standard from Bio-Rad laboratories (Hercules, CA, USA).
• Peak area , , r
• DoE Design of Experiments
• FIG. 3A-F present the DoE-generated prediction profiler showing the relationship between the tested factors temperature (panels A, D), pH, (panels B, E) and osmolality (panels C, F) within the chosen ranges on both aggregation (panels A, B, C) and final mAb titer (panels D, E, and F).
• the DoE prediction profiler indicates that to achieve less aggregation the cells should be cultured in medium with low osmolality and high temperature and pH. Within the chosen range of tested factors, the DoE prediction profiler indicates cells should be cultured in medium with starting osmolality of 320 mOsm/kg H 2 0 and cultured at pH 7.0 and 37°C. These conditions were predicted to lower mAb aggregation to only 1 .03% and increase final mAb concentration to 8.3 g/L.
• the DoE contour plots indicate cells should be cultured in medium with starting osmolality of 320 mOsm/kg H 2 0 and cultured at pH 6.85 and temperature of 36.5°C.
• Cell culture medium pH may influence the electrostatic interactions between mAb molecules by affecting the charge distribution on the protein surfaces.
• By lowering the system pH, moving further from the isoelectric point of the mAb it could be expected that increased charge densities would result in increased levels of like-charge repulsion between molecules. This in turn could be expected to reduce mAb aggregation.
• the results of the experiments performed in this study however showed that increasing the medium pH to 6.85 or 7.0, actually resulted in a reduction in mAb aggregation levels. Further reductions in mAb aggregation levels were observed when the cell culture medium pH was increased to a value of 7.2 (data not shown).
• Embodiment 1 A method of producing an anti-delta like ligand 4 (DLL4) monoclonal antibody, comprising:
• the antibody comprises:
• VH domain a heavy chain variable domain as set forth in SEQ ID NO:7 and a light chain variable (VL) domain as set forth in SEQ ID NO:8; or b) a VH domain complementarity domain region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:1 , a VH domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3; and a VL domain CDR1 comprising the amino acid sequence as set forth in SEQ ID NO:4, a VL domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 and a VL domain CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:6; and recovering the expressed anti-DLL4 antibody from the culture supernatant.
• CDR VH domain complementarity domain region
• Embodiment 2 A method of producing an anti-delta like ligand 4 (DLL4) monoclonal antibody, comprising:
• the antibody comprises: a) a heavy chain variable (VH) domain as set forth in SEQ ID NO:7 and a light chain variable (VL) domain as set forth in SEQ ID NO:8; or b) a VH domain complementarity domain region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:1 , a VH domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3; and a VL domain CDR1 comprising the amino acid sequence as set forth in SEQ ID NO:4, a VL domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 and a VL domain CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:6; and recovering the expressed anti-DLL4 antibody from the culture supernatant.
• VH heavy chain variable
• VL light chain variable
• CDR VH domain complementarity domain region
• Embodiment 3 The method of Embodiment 1 or 2 wherein the recovered anti-DLL4 antibody comprises less than 5% aggregate as determined by SEC-HPLC.
• Embodiment 4 The method of any of the preceding Embodiments wherein the recovered anti-DLL4 antibody comprises less than 2% aggregate as determined by SEC-HPLC.
• Embodiment 5 The method of any one of the preceding Embodiments further comprising feeding the cells with a two-part feed during the culturing.
• Embodiment 6 The method of any one of the preceding Embodiments, wherein the mammalian cell line is chosen from a Chinese Hamster Ovary (CHO), NSO, or PER.C6 cell line.
• Embodiment 7. The method of Embodiment 6, wherein the cell line is CHO.
• Embodiment 8 The method of any one of the preceding Embodiments, wherein the recovering of the anti-DLL4 antibody comprises affinity purification of the antibody.
• Embodiment 9 The method of Embodiment 8 wherein the affinity purification comprises protein A affinity chromatography.
• Embodiment 10 The method of any one of the preceding Embodiments wherein the titer of the antibody in the culture supernatant is at least 3 g/L.
• Embodiment 1 1 The method of any one of the preceding Embodiments wherein the titer of the antibody in the culture supernatant is at least 4 g/L.
• Embodiment 12 The method of any one of the preceding Embodiments wherein the titer of the antibody in the culture supernatant is at least 5 g/L.
• Embodiment 13 The method of any one of the preceding Embodiments wherein the titer of the antibody in the culture supernatant is at least 6 g/L.
• Embodiment 14 The method of any of the preceding Embodiments wherein the titer of the antibody in the culture supernatant is at least 7 g/L.
• Embodiment 15 The method of any of the preceding Embodiments wherein the anti- DLL4 antibody comprises VH domain as set forth in SEQ ID NO:7 and a VL domain as set forth in SEQ ID NO:8.
• Embodiment 16 The method of any of Embodiments 1 -14 wherein the anti-DLL4 antibody comprises a VH domain CDR1 comprising the amino acid sequence as set forth in SEQ ID NO:1 , a VH domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3; and a VL domain CDR1 comprising the amino acid sequence as set forth in SEQ ID NO:4, a VL domain CDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 and a VL domain CDR3 comprising the amino acid sequence as set forth in SEQ ID NO:6
• Embodiment 17 A method of reducing aggregate content in a protein A-purified monoclonal antibody product to less than about 5%, the method comprising:
• the cell line expresses an anti-DLL4 antibody comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1 , a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 6; and
• the culture process comprises using a two part feed to feed the cells; b) recovering the expressed antibody from the culture supernatant;
• Embodiment 18 A method of reducing aggregate content in a protein A-purified monoclonal antibody product to less than about 5%, the method comprising: a) culturing a mammalian cell line that expresses the antibody in a culture medium having a starting osmolality of about 320 mOsm/kg H 2 0, and at a temperature of about 36.5°C, and a pH of about 6.85;
• the cell line expresses an anti-DLL4 antibody comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1 , a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 6; and
• the culture process comprises using a two part feed to feed the cells; b) recovering the expressed antibody from the culture supernatant;
• Embodiment 19 The method of Embodiment 17 or 18 wherein the affinity chromatography comprises protein A affinity chromatography.
• Embodiment 20 The method of any of Embodiment 17-19 wherein the mammalian cell is a CHO cell.
• Embodiment 21 The method of any of Embodiments 17-20 wherein the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8.
• Embodiment 22 The method of any of Embodiments 17-21 wherein the purified anti- DLL4 antibody comprises less than 2% aggregate as determined by SEC-HPLC.
• Embodiment 23 The method of any one of Embodiments 17-22 wherein the titer of the antibody in the culture supernatant is at least 3 g/L.
• Embodiment 24 The method of any one of Embodiments 17-23 wherein the titer of the antibody in the culture supernatant is at least 4 g/L.
• Embodiment 25 The method of any one of Embodiments 17-24 wherein the titer of the antibody in the culture supernatant is at least 5 g/L.
• Embodiment 26 The method of any one of Embodiments 17-25 wherein the titer of the antibody in the culture supernatant is at least 6 g/L.
• Embodiment 27 The method of any of Embodiments 17-26 wherein the titer of the antibody in the culture supernatant is at least 7 g/L.
• Embodiment 28 A method of reducing aggregates of an anti-DLL4 monoclonal antibody (mAb) comprising culturing a CHO cell that secretes the anti-DLL4 mAb under conditions of temperature, pH, and osmolality, that produce less aggregate than culture of the same mAb-producing CHO cell under conditions comprising a temperature of 36.5°C, a pH of 6.8, and starting osmolality of 320 mOsm/kg H 2 O in a bioreactor using a single feed,
• mAb monoclonal antibody
• the anti-DLL4 antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1 , a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 6.
• Embodiment 29 The method of Embodiment 28 wherein the conditions that produce less aggregate comprise one of:
• Embodiment 30 The method of Embodiment 28 or 29 wherein the culturing further comprises feeding the cells with a two part feed.
• Embodiment 31 The method of any of Embodiments 28-30 wherein the anti-DLL4 antibody comprises a VH domain comprising the amino acid sequence as shown in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as shown in SEQ ID NO:8.
• Embodiment 32 An antibody composition produced by any of the preceding embodiments, wherein the antibody composition comprises less than about 1 .4% aggregate, as determined by SEC-HPLC.

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